CN109507368A - A kind of accurate Resistance Identification method of seedling stage sweet potato black rot - Google Patents
A kind of accurate Resistance Identification method of seedling stage sweet potato black rot Download PDFInfo
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- CN109507368A CN109507368A CN201811363796.1A CN201811363796A CN109507368A CN 109507368 A CN109507368 A CN 109507368A CN 201811363796 A CN201811363796 A CN 201811363796A CN 109507368 A CN109507368 A CN 109507368A
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- potato
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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Abstract
The present invention discloses a kind of accurate Resistance Identification method of seedling stage sweet potato black rot, belongs to agricultural plant protection field, including 1) germ separation and activation；2) preparation of PS culture solution；3) pathogen shakes training；4) preparation of alternaria spore suspension；5) potato seedling growth of participating in the experiment prepares and inoculation；6) research and appraisal；Identification method of the present invention is scientific and efficient, it is easy to operate, using spore liquid irrigating root inoculated identification, close to the actual conditions of field sweet potato's seedlings infection black spot, qualification result is more accurate reliable, potato seedling growth stalk is easy rotten problem during avoiding water planting potato seedling growth anti-disease enzyme simultaneously, the problem rotted is easy by living contaminants with using after potato wedge moisturizing during potato wedge progress anti-disease enzyme, a kind of accurate Resistance Identification method is provided for seedling stage sweet potato black rot, sweet potato black rot resistant variety, which is screened, for early stage rapid, high volume provides sound assurance, for instructing sweet potato breeding for disease resistance that there is important application value.
Description
Technical field
The present invention relates to a kind of accurate Resistance Identification methods of seedling stage sweet potato black rot, belong to agricultural plant protection field.
Background technique
Sweet potato black rot (sweetpotato blackrot) is one of the Major Diseases in sweet potato cultivation and storage, alive
There is generation in each sweet potato producing region in boundary, and pathogen is Ceratocystis fimbriata bacterium (CeratocystisfimbriataEllis
EtHalsted), can cause harm Sweet Potato, leaf and root tuber tissue, cause dead seedling, rotten bed, rotten cellar, very big to sweet potato Influence of production.Quilt
Containing substances such as ipomeamarones in the sick potato that alternaria infected, poisoning symptom can be caused after domestic animal is edible；With sick potato
Fermentation process can be delayed by making fermentation raw material, reduce alcohol output and quality, the serious development for restricting Sweet Potato Industry.Alternaria can
It is propagated by number of ways such as potato seed, seedling and soil, is difficult thoroughly to eradicate, producing upper breeding black spot disease-resistant variety at present is
One of main controlling way.
Anti-disease enzyme is the basis of breeding resistant variety, uses in production dip in spore liquid needle thorn potato wedge inocalation method and spore at present
Sub- liquid infects potato seedling growth method and carries out sweet potato variety black spot anti-disease enzyme, wherein being resisted using spore liquid needle thorn potato wedge inocalation method is dipped in
Disease identification takes around 11d, changes gauze daily and carries out moisturizing, the too small black spot of humidity is not susceptible in the process, humidity mistake
Big potato wedge is in that high humidity state is very easy to by living contaminants and potato wedge is easy to rot for a long time, seriously affects anti-disease enzyme knot
Fruit；Potato seedling growth method is infected using spore liquid and takes around 5-7d, potato seedling growth stalk is impregnated for a long time easily rots in a liquid, to disease-resistant
Qualification result affects greatly.Therefore, sweet potato black rot Resistance Identification is carried out using both methods to be lacking.
Therefore, a kind of accurate Resistance Identification method of sweet potato black rot is established, is this field technical problem urgently to be resolved,
It is the essential condition of sweet potato black rot breeding for disease resistance, and at present both at home and abroad there is not yet correlative study is reported.
Summary of the invention
In view of the above existing problems in the prior art, the present invention provides a kind of accurate Resistance Identification sides of seedling stage sweet potato black rot
Method, this method is easy to operate, scientific and efficient.
To achieve the goals above, the present invention use a kind of accurate Resistance Identification method of seedling stage sweet potato black rot, including with
Lower step:
1) germ separation and activation: morbidity potato wedge is acquired from field, is isolated and purified with tissue isolation and single spore separation method
Sweet potato black rot pathogen, and after the verifying of kock formula rule, using potato dextrose agar, cultivate 7 days, it is spare；
2) it the preparation of PS culture solution: weighs potato 20g addition 1L ultrapure water and boils to potato rotten soft, gauze mistake completely
15g sucrose is added after filter, adds ultrapure water to be settled to 1L, each triangular flask dispenses 30mL, then in 121 DEG C of high pressure sterilization 15min
It is spare；
3) pathogen shakes training: it activated sweet potato black rot pathogen is taken out, is beaten with the punch that diameter is 6mm and takes bacterium dish,
It is inoculated with 20 bacterium dish in each triangular flask, inoculated triangular flask is placed in 28 DEG C of constant-temperature tables, shakes training in 120rpm/min
48h；
4) preparation of alternaria spore suspension: filtrate is collected by filtration using filter paper, microscopically observation simultaneously adjusts spore
Sub- suspension concentration is 1 × 105CFU/ml；
5) potato seedling growth of participating in the experiment prepares and inoculation: taking sweet potato variety from sweet potato bunker, each 3 pieces, is placed in the basin alms bowl of dress soil
Vernalization, each kind clip 3 consistent sweet potato's seedlings of growth, are planted in respectively in the basin alms bowl of dress sterilized soil after germinateing, will
The spore suspension prepared carries out root irrigation, and each basin alms bowl pouring root amount is 50ml, is uniformly sprayed with aqua sterilisa within every 3 days later
Mist；
6) research and appraisal: potato seedling growth black spot incidence is investigated after inoculation 15 days, and will according to disease index grade scale
Potato seedling growth is divided into 0,1,3,5,7,9 grade, totally 6 grades, according to average disease index by resistance of the kind to black spot be divided into it is disease-resistant,
Four ranks of middle resistant, susceptible and high sense；Average disease index=(repeat 1 disease index+repetition, 2 disease index+3 state of an illness of repetition
Index)/3；Seedling stage sweet potato black rot resistance level is determined according to following standard:
It is disease-resistant: average disease index≤2.0；
In resist: 2.0 < are averaged disease index≤4.0；
Susceptible: 4.0 < are averaged disease index≤6.0；
Height sense: average disease index > 6.0；
The grade scale is:
0 grade: whole strain health is without illness；
1 grade: plant normally takes root, and underground part stem has a fragmentary blackspot, basal part of stem yellow leaf or has wilting, and overground part is normal
Branch growth；
3 grades: plant takes root but root system is less, and underground part stem has blackspot in flakes, basal part of stem yellow leaf, and potato seedling growth growth is delayed
Slowly；
5 grades: plant does not take root, and underground part stem has a blackspot in flakes, basal part of stem yellow leaf, potato seedling growth not branch completely；
7 grades: underground part necrosis is rotted, whole strain yellow leaf, and plant is wilted；
9 grades: underground part necrosis is rotted, and potato seedling growth lobus cardiacus rots, and whole strain is dead.
The beneficial effects of the present invention are:
1, party's forensic science is quick, step is simply easily operated, it is only necessary to culture dish, incubator, superclean bench, plastics
The conventional instruments consumptive material such as basin alms bowl, each breeding units can carry out identification work.
2, the present invention is inoculated into capable identification using alternaria spore suspension liquid irrigating root, and anti-sense phenotype is intuitive, avoids and dips in
Spore liquid needle pierces in potato wedge inocalation method, and potato meat is inconvenient the problem of observing and measuring blackspot transverse and longitudinal diameter, mirror in the case where purple sweet potato
It is relatively reliable to determine result.
3, the present invention uses alternaria spore suspension liquid irrigating root inoculated identification, infects blackspot close to field sweet potato's seedlings
The actual conditions of disease, qualification result are more accurate reliable；This method avoid dip in spore liquid needle thorn potato wedge inocalation method and spore simultaneously
Sub- liquid infects potato seedling growth method, and potato wedge and potato seedling growth are easy rotten problem in qualification process.The popularization and use of this method are big for early stage
Amount quickly screens disease-resistant material and sweet potato breeding for disease resistance is instructed to have important application value.
Detailed description of the invention
Fig. 1 is the flow chart that the present invention carries out seedling resistance identification using alternaria spore suspension liquid irrigating root；
Wherein, A figure is activated alternaria；B figure is the PS fluid nutrient medium prepared；It is 6mm that C figure, which is with diameter,
Punch beat and take bacterium dish spare；D figure is 20 bacterium dish of inoculation into PS culture medium；E figure is 28 DEG C, 120rpm/min shakes training
The mixed liquor obtained after 48h；F figure is the operation that spore liquid is collected by filtration using filter paper；G figure is to be collected by filtration and dilute through filter paper
To the spore suspension of respective concentration；H figure is to move the sweet potato seedling for plucking each kind after pouring root.
Specific embodiment
In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention is carried out below further detailed
It describes in detail bright.However, it should be understood that the specific embodiments described herein are merely illustrative of the present invention, it is not limited to this hair
Bright range.
Unless otherwise defined, all technical terms and scientific terms used herein are led with technology of the invention is belonged to
The normally understood meaning of the technical staff in domain is identical, and term as used herein in the specification of the present invention is intended merely to retouch
State the purpose of specific embodiment, it is not intended that in the limitation present invention.
Embodiment 1
As shown in Figure 1, a kind of accurate Resistance Identification method of seedling stage sweet potato black rot, specifically includes the following steps:
1) germ separation and activation: morbidity potato wedge is acquired from field, is isolated and purified with tissue isolation and single spore separation method
Sweet potato black rot pathogen, and after the verifying of kock formula rule, using potato dextrose agar, cultivate 7 days, it is spare；
2) it the preparation of PS culture solution: weighs potato 20g addition 1L ultrapure water and boils to potato rotten soft, gauze mistake completely
15g sucrose is added after filter, adds ultrapure water to be settled to 1L, each triangular flask dispenses 30mL, then in 121 DEG C of high pressure sterilization 15min
It is spare；
3) pathogen shakes training: it activated sweet potato black rot pathogen is taken out, is beaten with the punch that diameter is 6mm and takes bacterium dish,
It is inoculated with 20 bacterium dish in each triangular flask, inoculated triangular flask is placed in 28 DEG C of constant-temperature tables, shakes training in 120rpm/min
48h；
4) preparation of alternaria spore suspension: filtrate is collected by filtration using filter paper, microscopically observation simultaneously adjusts spore
Sub- suspension concentration is about 1 × 105CFU/ml；
5) potato seedling growth of participating in the experiment prepares and inoculation: 12 tested varieties are respectively C1: Nanjing 92；C2: Ji 982；C3: cigarette 25；C4:
Ji 26；C5:SQ16；C6: wide by 87；C7: Soviet Union 16；C8: fortune potato 66；C9: dragon 9；C10: quotient 19；C11: triumph hundred；C12: Xu's potato
29, each sweet potato variety is taken from sweet potato bunker, each 3 pieces, is placed in vernalization in the basin alms bowl of dress soil, each kind clip after germinateing
3 consistent sweet potato's seedlings of growth, are planted in respectively in the basin alms bowl of dress sterilized soil, and the spore suspension prepared is carried out pouring root
Handling each basin alms bowl pouring root amount is 50ml, and every 3 days later with aqua sterilisa even spraying；
6) research and appraisal: potato seedling growth black spot incidence is investigated after inoculation 15 days, and will according to disease index grade scale
Potato seedling growth is divided into 0,1,3,5,7,9 grade, totally 6 grades, according to average disease index by resistance of the kind to black spot be divided into it is disease-resistant,
Four ranks of middle resistant, susceptible and high sense；Average disease index=(repeat 1 disease index+repetition, 2 disease index+3 state of an illness of repetition
Index)/3；Seedling stage sweet potato black rot resistance level is determined according to following standard:
It is disease-resistant: average disease index≤2.0；
In resist: 2.0 < are averaged disease index≤4.0；
Susceptible: 4.0 < are averaged disease index≤6.0；
Height sense: average disease index > 6.0；
The grade scale is:
0 grade: whole strain health is without illness；
1 grade: plant normally takes root, and underground part stem has a fragmentary blackspot, basal part of stem yellow leaf or has wilting, and overground part is normal
Branch growth；
3 grades: plant takes root but root system is less, and underground part stem has blackspot in flakes, basal part of stem yellow leaf, and potato seedling growth growth is delayed
Slowly；
5 grades: plant does not take root, and underground part stem has a blackspot in flakes, basal part of stem yellow leaf, potato seedling growth not branch completely；
7 grades: underground part necrosis is rotted, whole strain yellow leaf, and plant is wilted；
9 grades: underground part necrosis is rotted, and potato seedling growth lobus cardiacus rots, and whole strain is dead.
Each Variety Disease-resistance qualification result is as shown in table 1.
1 sweet potato seedling stage black spot Resistance Identification result of table
As it can be seen from table 1 in 12 sweet potato varieties identified using the method for the present invention, equal totally a kind in Nanjing 92
(being) potato seedling growth is " disease-resistant " to the resistance rank of black spot；1 kind such as Ji 982 (is) that potato seedling growth is to the resistance rank of black spot
" in resist "；The resistance rank of totally 5 kind potato seedling growth black spots such as cigarette 25, wide 87, Soviet Union 16, fortune potato 66 and dragon 9 is " susceptible "；Ji
26, the resistance rank of SQ16, quotient 19, triumph No. hundred and Xu's potato 29 etc. totally 5 kind potato seedling growth black spots is " height sense ".
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modification, equivalent replacement or improvement etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (1)
1. a kind of accurate Resistance Identification method of seedling stage sweet potato black rot, which comprises the following steps:
1) germ separation and activation: morbidity potato wedge is acquired from field, isolates and purifies sweet potato with tissue isolation and single spore separation method
Alternaria, and after the verifying of kock formula rule, using potato dextrose agar, cultivate 7 days, it is spare；
2) preparation of PS culture solution: weigh potato 20g be added 1L ultrapure water boil to potato completely it is rotten soft, after filtered through gauze
15g sucrose is added, adds ultrapure water to be settled to 1L, each triangular flask dispenses 30mL, then spare in 121 DEG C of high pressure sterilization 15min；
3) pathogen shakes training: taking out activated sweet potato black rot pathogen, is beaten with the punch that diameter is 6mm and take bacterium dish, each
It is inoculated with 20 bacterium dish in triangular flask, inoculated triangular flask is placed in 28 DEG C of constant-temperature tables, shakes training 48h in 120rpm/min；
4) preparation of alternaria spore suspension: being collected by filtration filtrate using filter paper, microscopically observation and to adjust spore outstanding
Supernatant liquid concentration is 1 × 105CFU/ml；
5) potato seedling growth of participating in the experiment prepares and inoculation: sweet potato variety is taken from sweet potato bunker, each 3 pieces, is placed in vernalization in the basin alms bowl of dress soil,
The each kind clip 3 consistent sweet potato's seedlings of growth, are planted in the basin alms bowl of dress sterilized soil respectively, will prepare after germinateing
Spore suspension carry out root irrigation, each basin alms bowl pouring root amount is 50ml, and every 3 days later with aqua sterilisa even spraying；
6) research and appraisal: potato seedling growth black spot incidence is investigated after inoculation 15 days, and according to disease index grade scale by potato seedling growth
Be divided into 0,1,3,5,7,9 grade, totally 6 grades, according to average disease index by resistance of the kind to black spot be divided into it is disease-resistant, in
Four ranks of resistant, susceptible and high sense；Average disease index=(repeat 1 disease index+repetition, 2 disease index+3 state of an illness of repetition to refer to
Number)/3；Seedling stage sweet potato black rot resistance level is determined according to following standard:
It is disease-resistant: average disease index≤2.0；
In resist: 2.0 < are averaged disease index≤4.0；
Susceptible: 4.0 < are averaged disease index≤6.0；
Height sense: average disease index > 6.0；
The grade scale is:
0 grade: whole strain health is without illness；
1 grade: plant normally takes root, and underground part stem has a fragmentary blackspot, basal part of stem yellow leaf or has wilting, the normal branch of overground part
Growth；
3 grades: plant takes root but root system is less, and underground part stem has blackspot in flakes, basal part of stem yellow leaf, potato seedling growth slow growth；
5 grades: plant does not take root, and underground part stem has a blackspot in flakes, basal part of stem yellow leaf, potato seedling growth not branch completely；
7 grades: underground part necrosis is rotted, whole strain yellow leaf, and plant is wilted；
9 grades: underground part necrosis is rotted, and potato seedling growth lobus cardiacus rots, and whole strain is dead.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111187725A (en) * | 2020-03-17 | 2020-05-22 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Efficient separation method of sweet potato shell-rostratum |
| CN111264254A (en) * | 2020-02-28 | 2020-06-12 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato southern blight resistance identification method |
| CN111418382A (en) * | 2020-03-31 | 2020-07-17 | 河南省农业科学院植物保护研究所 | Method for nondestructive inoculation of alternaria alternata on sweet potato blocks |
| CN112014524A (en) * | 2020-09-04 | 2020-12-01 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | Method for identifying potato scab resistance |
| CN115039588A (en) * | 2022-05-17 | 2022-09-13 | 新疆农业科学院综合试验场 | Method for identifying black nevus disease resistance of potato cutting seedlings |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111264254A (en) * | 2020-02-28 | 2020-06-12 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Sweet potato southern blight resistance identification method |
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| CN111418382A (en) * | 2020-03-31 | 2020-07-17 | 河南省农业科学院植物保护研究所 | Method for nondestructive inoculation of alternaria alternata on sweet potato blocks |
| CN112014524A (en) * | 2020-09-04 | 2020-12-01 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | Method for identifying potato scab resistance |
| CN112014524B (en) * | 2020-09-04 | 2022-07-08 | 宁夏农林科学院农业生物技术研究中心（宁夏农业生物技术重点实验室） | Method for identifying potato scab resistance |
| CN115039588A (en) * | 2022-05-17 | 2022-09-13 | 新疆农业科学院综合试验场 | Method for identifying black nevus disease resistance of potato cutting seedlings |
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